Our prior work has focused on the molecular mechanisms underlying Drosophila's circadian rhythms. Orthologs of genes initially characterized in Drosophila have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. Here we propose three classes of interdisciplinary investigations of the fly's rhythmic activity/rest behavior. (1) We wil use elav-Gal4 to drive individual, UAS-RNAi transgenes from two extensive libraries in a search for novel genetic pathways regulating sleep. Our objective is to test the full complement of Drosophila's protein-coding genes using ~19,000 RNAi lines. (2) We will extend our studies of a novel, reduced-sleep mutant, insomniac. We will determine whether insomniac regulates sleep in an active/dynamic manner, or whether it regulates a developmental pathway that is essential for wild type levels of sleep. We will test the hypothesis that Insomniac functions as a substrate-specific adapter for the Cul3 ubiquitin ligase complex, and will use a variety of biochemical and molecular approaches to identify a target substrate(s). (3) We will further define the role of a small group of cell cycle genes, CycA, its regulator (Rca1), cdk2 and cdc42, in the regulation of sleep. As constitutive activation of a cluster of neurons expressing cell cycle genes induces excess sleep, we will isolate these cells by flow cytometry to discover patterns of gene expression that may underlie the contribution of these cells to sleep. PUBLIC HEALTH RELEVANCE: The pressing need to understand sleep is underscored by the major clinical and economic impact of sleep disorders~ 10-15 percent of the U.S. population suffers from chronic insomnia, and nearly one hundred sleep disorders have been described, including narcolepsy, sleep apnea, and delayed and advanced sleep phase syndromes. Molecular genetic studies that began in Drosophila have already allowed mutant orthologs of Period protein and casein kinase 1 to be connected to inborn errors of human sleep. We believe our proposed genetic, cellular, and biochemical studies of Drosophila's activity/rest behavior will continue to provide insights and tools relevant to the understanding of human sleep.